TITLE:
Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Liver of Golden Cuttlefish Sepia esculenta
AUTHORS:
Ryo Nishino, Hiromi Kakizaki, Hideto Fukushima, Masahiro Matsumiya
KEYWORDS:
Chitinolytic Enzyme, Chitinase, Distribution, cDNA Cloning, Golden Cuttlefish Sepia esculenta
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.8 No.10,
October
13,
2017
ABSTRACT: The
distribution of chitinolytic enzymes in eight organs of the golden cuttlefish Sepia esculenta was
determined. Chitinase activity (activity of endo-type chitinolytic enzyme) was
measured using pNP-(GlcNAc)n (n = 2, 3) as substrates, with high activity detected in the liver, posterior
salivary gland, and stomach. β-N-acetylhexosaminidase
(Hex) activity (activity of exo-type chitinolytic enzyme) was determined using pNP-(GlcNAc)
as a substrate, and high activity was observed in six organs, including the
liver, branchial heart, posterior salivary gland, and stomach. In addition, two
chitin-binding proteins (CBP-A, CBP-B) were isolated from the liver using a
chitin affinity column. Two full-length cDNAs (SeChi-1: 1484
bp; SeChi-2: 1748
bp) encoding chitinases were obtained from the liver of S. esculenta. SeChi-1
contained a 1377-bp open reading frame (ORF) encoding 459 amino acids, and SeChi-2
contained a 1656-bp ORF encoding 552 amino acids. Domain structures predicted
from the deduced amino acid sequences of SeChi-1 and SeChi-2
(SeChi-1, SeChi-2) contained signal peptides, a GH Family 18 catalytic domain,
one chitin binding domain (CBD) in SeChi-1, and two CBDs in SeChi-2. Proteome
analysis revealed that 125 peptide residues of CBP-A were present in SeChi-1,
and 116 peptide residues of CBP-B were present in SeChi-2. Organ expression
analysis revealed that SeChi-1 and SeChi-2 were
expressed only in the liver of S. esculenta.
Phylogenetic analysis of SeChi-1, SeChi-2, and GH family 18 chitinases revealed
that SeChi-2 belongs to a group of previously reported squid chitinases, while SeChi-1 does not belong
to any previously reported group of mollusk chitinases.